Cryptococcus neoformans factors contributing to penetration of the blood-brain barrier Abstract Cryptococcus neoformans causes life-threating central nervous system (CNS) infection in immunocompromised individuals such as HIV-1-infected patients, resulting in over 180,000 deaths annually. Infection with C. neoformans starts with inhalation of fungal cells from environment, followed by extrapulmonary spread, which leads to hematogenous dissemination to target organs, most commonly resulting in CNS infection. Several lines of evidence of human cases and experimental animal models of C. neoformans CNS infection indicate that circulating C. neoformans penetrates into the brain initially via the cerebral capillaries. Since C. neoformans entry into the brain occurred in the cerebral microvasculature, we and others used the in vitro blood-brain barrier model with human brain microvascular endothelial cells (HBMEC) to investigate C. neoformans penetration of the blood-brain barrier. We showed that C. neoformans strains exhibited the ability to traverse the HBMEC monolayer in vitro and penetrate into the brain in vivo in the mouse model of experimental hematogenous C. neoformans CNS infection, but the underlying mechanisms remain incompletely understood. We and others showed that C. neoformans exploits cryptococcal factors for penetration of the blood-brain barrier, but determination of such factors remains incomplete. We generated genome-scale sgRNA library from C. neoformans genomic DNA. Each sgRNA cassette serves as a genetic barcode for mutation tracking, which allows high-throughput screen by deep sequencing. We hypothesize that this library allows genome-wide screen of C. neoformans factors contributing to penetration of the blood-brain barrier. This hypothesis is supported by our identification of cryptococcal factors using our in vitro (transcytosis assays in HBMEC monolayer) and in vivo blood-brain barrier models (animals models of cryptococcal penetration into the brain following intravenous and intranasal inoculations). Our proof of concept study with two knock-out mutants demonstrated that such C. neoformans factors contributed to penetration of the blood- brain barrier in vitro and in vivo. These findings suggest that C. neoformans factors identified from our genome- wide screen method are likely to contribute to penetration of the blood-brain barrier, the essential step in the development of C. neoformans CNS infection. The innovative aspect of this application is to investigate how C. neoformans penetrates the blood-brain barrier by elucidating newly identified cryptococcal factors. The information derived from this application will provide a new paradigm for investigating the pathogenesis of C. neoformans CNS infection.